The publications and other materials referred to herein to describe the background of the invention and to provide additional detail with regard to the practice of this invention are incorporated herein by reference.
Interferons are proteins that are secreted from cells in response to a variety of stimuli. Interferons are classified as Type I and Type II, depending on the cell receptor to which they bind. Type I consists of seven classes, including interferon alpha, which is produced by human leukocytes, and interferon beta, which is produced by fibroblasts. Type II consists only of interferon gamma. Type I interferons exhibit a wide breadth of biological activity, including antiviral, anti-proliferative, neoplastic and immunomodulatory activities. Therefore, they are useful in the treatment of a variety of diseases, including many viral diseases, such as viral hepatitis, and several cancers, such as hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia and metastatic malignant melanoma.
Human interferon was first isolated in 1957 by Isaacs and Lindenmann. Isaacs A. and Lindenmann J., “Virus interference. I. The interferon,” Proc. R. Soc. Lond. Ser. B. Biol. Sci. (1957) 147: 258-267. Many years later, interferon cDNAs from a virus-induced myeloblast cell line were analyzed, revealing the presence of many distinct species of interferon. Although analysis of this cDNA revealed differences in amino acid sequences, all reports suggested that active human leukocyte interferons (interferon alpha) had 165 or 166 amino acids. Levy, however, reported that a significant fraction of active interferon isolated from human leukocytes lacked the ten carboxy-terminal amino acids suggested from the DNA sequence of such interferons. See Levy, W. P., et al., “Amino acid sequence of a human leukocyte interferon,” Proceedings of the National Academy of Sciences (1981) 78(10): 6186-6190. In addition, Levy reported that this C-terminal truncation did not affect the specific activity of these proteins, thus indicating that the 10 COOH-terminal amino acids were not essential for interferon activity. See id. at 1689.
Nevertheless, bacterially produced recombinant interferon alpha (2a and 2b), which was approved for therapeutic use in 1986, has 165 amino acids. Researchers have attempted to enhance the biological activity of interferon alpha through modifications to the internal amino acids of the interferon rather than via carboxy terminal truncations. See, for example, Ozes, O. N., et al., “A comparison of interferon-con1 with natural recombinant interferons-α: antiviral, antiproliferative, and natural killer-inducing activities,” Journal of Interferon Research (1992) 12:55-59.
The present invention relates to the surprising discovery that recombinant interferon alpha that is truncated at the carboxy terminus exhibits enhanced biological properties compared to full length interferon. Applicants made this discovery while conducting experiments aimed at optimizing expression of full length interferon alpha protein in plants. Such plant-produced protein demonstrates anti-viral and anti-proliferative activity comparable to bacterially produced interferon alpha but contains C-terminal truncations that predominantly occur during processing of the plant material. A purification process was devised that reduced the carboxy terminal truncations to approximately 4% of the total interferon product but resulted in substantial loss of the desired product during processing. To obtain better yields and a more homogeneous product, Applicants prepared recombinant interferon alpha polypeptides lacking 1-9 of the C-terminal amino acids of full length interferon and found that these polypeptides displayed enhanced biological activity and enhanced processing qualities.